Inhibitory Effect Of Curcumin On Proliferation Of Human Lens Epithelial Cells

Objective Curcumin(CUR) is phenol pigment extracted from the rhizome ofCurcuma longa, which has been used to treat inflammatory disorders and cancer, butthe mechanism remains unclear. It may be related to the inhibition of proliferationand the induction of apoptosis. Residual lens epithelial cells proliferate in lenscapsule after cataract surgery, resulting in after cataract, which directly causes visualdisturbances. This experiment is to investigate the inhibitory effects of curcumin onproliferation of human 1 ens epithelial cell line SRA01/04(HLEC SRA01/04), and itsdose-dependent, time-dependent manner.Methods (1) Cell culture: HLEC were cultured in DMEM medium supplementedwith 10%(v/v) fetal calf serum(FBS). It was incubated at 37℃in 5% CO₂ gasincubator with saturation humidity. (2) MTT assay: Cells in exponential growth phasewere seeded into 96-well plates at a density of 8×10⁴/ml in 100ul tissue culture perwell, HLEC were incubated with CUR at different concentrations(0, 5, 10, 20, 30mg/L)and 0.1%DMSO for different hours(12h, 24h, 48h), then the absorbance was measuredwith a multiscanner autoreader and then calculate the inhibition ratio of growth. (3)Flow cytometric analysis(FCM): Assay the apoptosis of cell by APO2.7 after HLECwere incubated with CUR at different concentrations(0, 5, 10, 20, 30mg/L) for 24h. (4)Cell morphologic change: After treatment with CUR for 24h, HLEC morphologicchange was observed under inverted phase contrast microscope. At the same time,HLEC were washed twice with phosphate-buffered saline(PBS) and mixed with DAPI at a final concentration of 0.1mol/L, after 30 min of staining, cellular nuclearchange was observed under fluorescence microscope. (5) Immunohistochemistry:After treatment with CUR for 24h, the expression level of caspase-3 was performedby immunostaining analysis.Results (1) MTT assay: CUR can inhibit the growth of HLEC obviously. Bystatistical analysis, on 12h and 24h, the inhibition ratio of growth is distinct in groups(20mg/L, 30mg/L); on 48h, the inhibition ratio of growth is distinct ingroups(10mg/L, 20mg/L, 30mg/L). The difference is significant by statisticalanalysis(P＜0.05). The inhibition ratio of growth ascended on 24h in 5mg/L and10mg/L groups, after 24h, difference isn't obvious. The inhibition ratio of growthascended after 24h in 20mg/L group, and continued ascending. The inhibition ratioof growth ascended after 48h in 30mg/L group, before 48h, difference isn't obvious.The inhibitory effect of CUR on HLEC is markedly correlated with the dose. On 12h,24h and 48h, the inhibition ratio of growth isn't distinct in 0.1%DMSO group, thedifference isn't significant by statistical analysis(P＞0.05). (2) FCM assay: Althoughapoptosis can be found in control, group, the ratio of apoptosis is very low. Theapoptosis of cell was obviously observed in the treated groups, and the ratioascended depending on the dose. The difference is significant by statisticalanalysis(P＜0.05), but the difference between control group and 5mg/L group isn'tsignificant(P＞0.05). (3) Cell morphologic change: After treatment with CUR atdifferent concentrations for 24h, characteristic apoptotic features such as chromatincondensation, nuclear fragmentation, cellular shrinkage and apoptotic bodies wereexamined under inverted phase contrast microscope and fluorescence microscope. (4)Immunohistochemistry: After treatment with CUR at different concentrations for 24h,caspase-3 expression increased depending on the dose. Comparing with controlgroup, the difference is significant by statistical analysis(P＜0.05), but the differencebetween 20mg/L and 10mg/L group is not significant(P＞0.05). Conclusion CUR can inhibit proliferation and induce apoptosis in HLEC at adose-dependent and time-dependent manner. CUR may become a potential drug toprevent and cure after cataract.